High cis beta-carotene composition

ABSTRACT

A carotenoid composition derived from a natural source wherein at least 50% by weight of the carotenoid content of the composition is cis beta-carotene and preferably 9 cis beta-carotene. Typically, the beta-carotene content of the composition is predominantly 9 cis beta-carotene.

FIELD OF THE INVENTION

The invention relates to a carotenoid composition derived from a naturalsource, with a high cis beta-carotene concentration and its preparationfrom natural sources, and more preferably a high 9 cis beta-carotenecomposition and its preparation from natural sources.

BACKGROUND OF THE INVENTION

In this specification it is to be understood that the natural sources ofcarotene include fruits, vegetables and other plant tissue, and animaltissue. A particularly important commercial source of carotene iscertain types of algae.

Beta-carotene occurs in a number of different chemical isomer forms.Some of these are geometrical isomers which have a different orientationaround one of the double bonds In the conjugated double bond structureof the molecule.

This can occur in a number of positions along the conjugated backbone tomake a range of different geometrical isomers. In some cases there caneven be more than one double bond where change of orientation occurs.

The most common geometrical isomer is the all trans isomer with astructure occurring as shown as follows where the main carbon chain ofthe molecule occurs in a trans (across) or straight configuration.##STR1##

However, there are cis forms of beta-carotene which occur naturally, andcan be produced by chemical synthesis, or formed by physical processeslike heat on the all trans isomers, where the main carbon chain of themolecule takes a bend (cis) or sideways configuration. Naturallyoccurring cis forms of beta-carotene are not known to occur over aweight percentage of approximately 30% to less than 50% of the totalcarotenoid content.

Associated with the different geometric isomers are different propertiesand possible functions and for this reason there are potential benefitsin relatively concentrated forms of the cis isomers.

In natural products such as fruit, vegetables, algae and other plant andanimal material the carotenoids are stabilized as part of the cellstructure in small micron or sub-micron sized particles in the cellorganelles or even by association with other molecules which stabilizethe isomeric forms produced by the biochemical pathways of the organism.However, in the preparation of concentrated forms of these materials forcommercial products desired from the natural sources, the naturalstabilising capacity of the cellular structure may be removed or reducedin the extraction and concentration of the carotenoids.

In addition, as the carotenoid products are concentrated to increasetheir beta-carotene concentrations and to remove the other cell materialwhich is not desired in the product, there is a natural tendency forcertain isomers to crystallize out.

Crystallisation is a problem in certain applications since thecrystalline form may not be available for efficient use in theapplication because of its relative insolubility. Crystallisation occursparticularly with all trans beta-carotene and as a result it is not, forexample, readily available for biological use.

Cis isomers, on the other hand are much less likely to crystallise andas a consequence are much more soluble than the trans isomers. For thisreason, it is often more desirable to use beta-carotene containingcompositions with higher concentrations of cis isomers for variousapplications. For example, the 9 cis isomer is much more readily solublein oils than the all trans form. In fact, it is very difficult to getthe 9 cis isomer to crystallize out from naturally derived oils, thusmaking it difficult or expensive to purify on a large scale.

Unlike trans isomers, cis isomers have a number of applications,including use in water dispersible food colourants and in tablettingpowders. The cis isomers are also useful in a water dispersible form asemulsions for colouring and in beverages. They can also be used inspecial vitamin supplements in concentrates for direct supplementationor as part of food.

In naturally occurring products the proportion of cis isomers is rathersmall, but one of the highest proportions occurs in the halophillic algaDunaliella salina where normally 30% to below 50% of the totalcarotenoid content occurs as the 9 cis form.

The present invention is accordingly directed to compositions with ahigh cis beta-carotene composition derived from natural sources.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a sample chromatogram displaying the isomericanalysis of a carotenoid composition as described in the Examples.

DESCRIPTION OF THE INVENTION

Accordingly, in one form of this invention, a carotenoid compositionderived from a natural source is produced, wherein at least 50% byweight of the carotenoid content of the composition is cis beta-caroteneand preferably 9 cis beta-carotene. Typically, the beta-carotene contentis predominantly 9 cis beta-carotene.

In another form of the invention, the preferred range of cisbeta-carotene is between at least 50% and 80%, and more preferablybetween 60% and 70%. Another preferred range is between 60% and 85%.

In yet another preferred form of the invention, the high cisbeta-carotene from natural sources is derived (for example, byconcentration and purification by physical means) from a product oflower concentrations of the cis beta-carotene to a concentration of atleast 70% cis beta-carotene. Preferably this may be achieved by theremoval of the substantially all trans beta-carotene using physicalprocesses.

These high percentage cis isomer compositions have been found to exhibithigh solubility and are readily available for physiologically activepurposes. This is thought to be because of the following factors inparticular:

(a) cis isomers occur in human body tissues in significant amounts andsince the action of metabolism occurs in the tissues it is possible thatthe cis isomers have a physiological function;

(b) cis isomers are typically lower in the bloodstream which is higherin the all trans isomer; this suggests the cis isomers may be rapidlytaken into the tissues; and

(c) cis isomers appear to be easily absorbed from the intestine.

For example, high percentage cis isomers would be expected to haveimproved effect in use in medical applications, for example, in thehealing and prevention of cancer, cardiovascular disease and otherillnesses, since the cis isomers, because of their solubility, arelikely to preferentially accumulate in the tissues being removed fromthe bloodstream over the trans form.

The improved solubility is also likely to assist in the application ofthe high percentage cis isomer in the topical application of thecomposition and for ease in the preparation in food colour applications.The cis isomers may assist in the stability of emulsions or powders ofbeta-carotene for commercial purposes which is partly due to theirsolubility characteristics.

The structure of the 9 cis isomer of beta-carotene is as follows:##STR2##

This 9 cis isomer of beta-carotene is preferably derived from particularnatural sources of plant products including green peppers, apricots,flowers of certain species of the Acacia genus, cucurbitaceae and in thealga, Dunaliella salina, which has the highest concentration of the 9cis isomer of such sources. In this regard, see Ami Ben-Amotz, AmnonLers and Mordhay Aron: "Steroisomers of Beta-Carotene and Phytoene inthe Alga Dunaliella bardawil" Plant Physiol. (1988) 86, 1286-1291(Dunaliella bardawil has subsequently been acknowledged by Ami Ben-Amotzas naturally occurring Dunaliella salina).

The proportion of the total cis isomers (predominantly the 9 cis isomer)content in the total carotenoid content of the alga Dunaliella salina isnormally found at around 30% to below 50% of the total carotenoidcontent on a weight basis as determined by the high pressure liquidchromatography (see method below commencing at page 14) and visiblelight spectrophotometry techniques.

Preferably, the increase of the 9 cis isomers in the composition is toabout 70% of the total carotenoid absorbance and more preferably 80% ofthe total carotenoid content. The remaining 20% typically would consistof all trans beta-carotene (for example 10%), other cis forms (forexample 5%) and other carotenoids (for example 5%).

The finished product is preferably dispersed in a natural carrier oilfrom animal, vegetable and mineral origins and particularly olive, corn,soya bean, essential oils, terpene based oils and fish derived oils.

The provisions of small quantities of oil soluble anti-oxidants may bebeneficial for certain applications of the product. Examples of thesewould be butylated hydroxy anisole, butylated hydroxytoluene, propylgallate, ethoxyquin and ascorbyl palmitate plus other naturalantioxidant extracts, for example, derived from herbs and preferablynatural tocopherols.

The anti-oxidants may be used to assist to protect the high cisbeta-carotene preparations from oxidation, which is relatively moreimportant when in a lower total beta-carotene concentration product, forexample, less than 5% of beta-carotene in the preparation. However, evenat higher concentrations it is important to protect the beta-carotenepreparations from oxidising.

A concentration of 0.01% to 1.0%, preferably 0.01% to 0.5%, of the pureactive anti-oxidant is typically used, depending on the actualanti-oxidant of choice and application, as this concentration range hasbeen shown by experience to be sufficient for normal protection of theultimate composition over its shelf life.

EXAMPLES OF METHODS TO PRODUCE THE HIGH CIS ISOMER COMPOSITION

The product is prepared by a series of physical unit operations.Examples of methods used to produce high cis isomer compositions, andmore preferably to produce high 9 cis isomers are set out below asExample A and Example B.

Example A demonstrates a method using centrifugation (which relies onthe principle of separation by density difference). The crystallinefraction containing the trans crystal is separated from the cis isomersoluble oil fraction.

This type of separation can either be done in a batch type centrifuge,usually on a small scale or for laboratory use or in a continuousmachine to process larger volumes on an industrial scale.

The density of the all trans isomer crystals is greater than the cisisomers (including the 9 cis isomer) when in solution in vegetable oilshaving a relative density of about 0.92 grams per cubic centimeter. Whenthe temperature or melting point of the carrier vegetable oil permitsthe oil to be liquid and of a viscosity to allow the all trans crystalsto settle when the centrifugal force is applied, centrifugation willseparate the all-trans isomer from the cis isomers. Being of the greaterdensity, the all trans crystals can be separated by sedimentation andleave the machine in the heavy fraction and the cis isomers stay in thesoluble vegetable oil or light fraction.

Centrifuges (for example, an Alfa Laval model number FUVPX207) which arecapable of opening the bowl to remove a semi solid heavy sludge canconcentrate the all trans crystals in this sludge thus increasing theyield of the high cis light fraction. The flow rate of material throughthe centrifuge has a large bearing on the result so this has to beoptimized for the machine concerned.

Example B relies on filtration using a basket type centrifuge and filterbag, however, it will be readily understood by a person skilled in theart that the filtering operation can be performed on a range of filtermaterials, including a filter press, drum filter, filter beds, filtercartridge systems and more sophisticated membrane type filters. Anyfilter system can be used that employs pads, paper, cartridge filters ora centrifugal filtration using a filter in a basket centrifuge.

The crystals of all trans beta-carotene are held by the filter materialbut the soluble all cis material can pass through the filter thusincreasing the proportion of cis isomers in the filtrate, that is, theproduct passing through the filter material.

Example A.

Ten grams of 4% natural beta-carotene in soya bean oil (commerciallyavailable from Betatene Limited and sold under the brand name "BetateneLimited 4% Natural Beta-Carotene in Soya Bean Oil") was placed in an 18mm diameter glass centrifuge tube and centrifuged at 2000 revolutionsper minute for 15 minutes at 21° C. in a Clements 2000 laboratorycentrifuge.

At the conclusion, the supernatant oil (the light fraction) wasrecovered by removal with a Pasteur pipette and assayed for cis andtrans beta-carotene content. After draining the residual supernatantfrom the heavy fraction, the same analysis was performed on the pelletedheavy fraction.

The percentages of the cis (including the 9 cis) beta-carotene and alltrans beta-carotene (determined using the the high pressure liquidchromatography method commencing on page 14) were as follows:

    ______________________________________                                                    CIS total All TRANS                                                           predominantly 9 cis                                                           % of total carotenoid by weight                                   ______________________________________                                        Original 4%   42          45                                                  Light fraction                                                                              62          24                                                  Heavy fraction                                                                              19          74                                                  ______________________________________                                    

with the crystals being collected in the heavy phase or the sludgefraction and the high cis product as the light fraction. The residueincludes other carotenoids.

The same procedure can be performed on a continuous production scaleusing an open bowl desludging centrifuge such as an Alfa Laval modelnumber FUVPX 207. The flow rate of the feed to the centrifuge, thesetting of the skimmer to recover the high cis light fraction and thedesludging timing would need to be optimised for the machine or othersimilar machines. This will be well understood by persons skilled in theart.

Example B.

A crystalline suspension of beta-carotene in soya bean oil containing21% total carotenoid was filtered to retain crystals and to allow thesoluble fraction to pass through. The all trans crystals can beseparated from an oil based matrix, providing the oil is not too viscousto restrict movement of the oil through the filter bed. The startingmaterial is available from Betatene Limited and is sold under the brandname "Betatene Limited 20% Natural Beta-Carotene in Soya Bean Oil".

This operation can be achieved in a basket type centrifuge of a typelike a Broadbent, Tolhurst for Burton with a 900 mm diameter bowl, or asimilar machine. The bowl is made of perforated stainless steel plateand a fabric bag is placed in the bowl with a weave having gaps betweenthe fibres of approximately 20 to 25 microns in width. The 20% naturalbeta-carotene in soya bean oil is fed into the bag and the high transcrystals are retained on the bag letting the soluble high cis fractionpass through the bag and the perforations in the bowl. In the initialstages the filtrate may still contain the smaller crystals but this isovercome by recirculation through the centrifuge. Before long thecrystal bed on the inner surface of the bag acts as the main filterwhich will remove the small crystals. The operation continues till theflow of filtrate through the bag is too slow. The filtrate is collectedas are the retained crystals at the end of the run and analysed forbeta-carotene and cis-trans isomeric profile.

The percentages of the cis beta-carotene and all trans beta-carotene(determined using the using the the high pressure liquid chromatographymethod commencing on page 14) were as follows:

    ______________________________________                                                      CIS       TRANS                                                               predominantly 9 cis                                                           % of total carotenoid by weight                                 ______________________________________                                        Initial 21% oil suspension                                                                    32          63                                                Filtrate        82          14                                                Filtered crystals                                                                             27          68                                                ______________________________________                                         The residue includes other carotenoids.                                  

Example A is an actual example of a method that has been carried out ona laboratory scale. Example B was carried out on a production scale.

It would also be understood by persons skilled in the art that cisisomers could be separated on a larger scale using preparative highpressure liquid chromatography. For example, Dunaliella salina algaewhich has a 9 cis isomer content of below 50% could be treated bypreparative high pressure liquid chromatography techniques to separatethe isomers and obtain a very high purity product of the 9 cis isomer(for example, 80% or more of the 9 cis isomer). This separationtechnique relies on the variable retention of different chemicalmaterials to a solid phase when the materials in various mixtures ofvolatile organic solvents are pumped through the solid phase, usually inthe form of spheres in a column. By collecting fractions at the end ofthe column the chemical materials may be separated from each other. Thevolatile organic solvents are evaporated from the pure chemical toprovide the solvent free product.

EXAMPLE OF ISOMERIC ANALYSIS OF A CAROTENOID COMPOSITION USING HIGHPRESSURE LIQUID CHROMOTOGRAPHY

The following is an example of a standard method for conducting anisomeric analysis of a carotenoid composition using high pressure liquidchromatography to determine the percentage content of the cis isomers ofbeta-carotene and all trans beta-carotene. This was the method used inanalysing the isomeric percentages in Example A and Example B above.

In summary, a sample of a beta-carotene containing composition in oil isdissolved in cyclohexene and diluted to a suitable concentration. Beforeinjecting the resultant solution into the high pressure liquidchromatograph, the sample should be diluted with a mobile phase. Theconcentration of beta-carotene is determined by obtaining an absorbanceat a specific wavelength using a known extinction coefficient. The cisand trans contents are determined by separating the isomers by highpressure liquid chromatography. The percentages of cis isomers and transisomers are then determined as a percentage of the total carotenoidcontent.

(i) Reagents and Equipment

The following is a list of the reagents and equipment that can be usedin the analysis.

Spectrophotometer with 10 mm glass cells

Analytical balance

Cyclohexane AR grade

100 ml volumetric flasks

50 ml volumetric flasks

2.0 ml bulb pipettes

Chloroform AR grade

High pressure liquid chromatograph incorporating:

Isocratic pump

Injector able to handle up to 50 μl

UV/Vis detector set at 453 nm

Vydac 201TP54 reverse phase column 250 mm

Acetonitrile HPLC grade

Methanol HPLC grade

10 ml volumetric flask

(ii) Preparation of the mobile phase

Prior to the analysis being conducted, a mobile phase should be preparedby weighing 400 g of acetonitrile into a 1000 ml storage bottle, adding300 g methanol and mixing well. The resultant mixture should be adjustedto room temperature before being used in the analysis.

(iii) Procedure

The method used in the actual analysis is set out below.

A sample of beta-carotene in oil is weighed accurately (to within 0.01g) to the equivalent to 80 mg of beta-carotene into a 100 ml volumetricflask. Approximately 5 ml of chloroform is added and mixed well untilthe sample has dissolved. To ensure that the sample has completelydissolved it should be viewed against a light source. If the sample isnot completely dissolved, it should be allowed to stand forapproximately 5 minutes. It is also possible to add a further 5 ml ofchloroform and to warm the mixture under, for example, hot tap water.

The volume of the mixture is then diluted with cyclohexane and mixedwell. This is solution A. 2 ml of solution A is then pipetted into a 50ml volumetric flask and diluted to volume with cyclohexane and mixedwell. This is solution B. 2 ml of solution B is then pipetted into a 50ml volumetric flask and diluted to volume with cyclohexane and mixedwell. This is solution C. The absorbance of solution C is then measuredat 455 nm against a cyclohexene blank (this gives the totalbeta-carotene content and not the isomeric ratio). 1 ml of solution B isthen pipetted into a 10 ml volumetric flask, diluted to volume with thecarrier solvent and mixed well. This is solution D.

(iv) Standards

For comparative purposes, a standard is then prepared. The standard isprepared to provide a reference material of known concentration in theassay and to determine reference retention time ("R.T.") for the alltrans isomer. The standard is made up according to the same procedureused to make up the sample. Weigh the reference standard material (Sigmaall trans beta-carotene from Sigma Corporate in St Louis USA) to theequivalent of 80 mg of beta-carotene, then proceed as for the sample.The concentration of the standard is determined in the same way as thesample.

(v) Determination

The standard and the sample as prepared above are ready for injection.

The flow rate of the high pressure liquid chromatograph should be set to1.0 ml/minute, 20 μl of the standard is injected into the high pressureliquid chromatograph and allowed to run for 25 minutes. The injectionconcentration and volume are recorded. 20 μl of solution D is theninjected into the high pressure liquid chromatograph and allowed to runfor 25 minutes. The injection concentration and volume are recorded.

An example of a typical chromatogram is reproduced in FIG. 1.

(vi) Calculation

I--Calculation for Determination of Beta-carotene Concentration

The bracketed figures are typical figures.

    ______________________________________                                        Absorbance of solution C at 455 nm                                                                  Abs     (-0.4)                                          Weight of sample      m       (-0.5 g)                                        Extinction coefficient E %                                                                          2500    cm.sup.-1 %.sup.-1                              Injected concentration (mg/l)                                                                       I       (-4)                                            ______________________________________                                         I = Abs × 25 × 10,000/(2,500 × 10) = Abs × 10    

II--Calculation for Determination of Cis and Trans Percentages

    ______________________________________                                        Peak area of all-trans beta-carotene                                                             T     (17%) R.T.- 13.6 minutes                             Peak area of unknown cis isomer 1                                                                C1    R.T.- 14 minutes (not                                                         seen in above                                                                 chromatogram)                                        Peak area of unknown cis isomer 2                                                                C2    (9.8%) R.T.- 14.7                                                             minutes                                              Peak area of 9-cis isomer                                                                        C3    (43%) R.T.- 15.5 minutes                             Peak area of 15-cis Isomer                                                                       C4    (8%) R.T.- 16.68 minutes                             ______________________________________                                    

The retention times used in the above table are the retention times thatwere seen in the chromatogram shown in FIG. 1. In the chromatogram shownin FIG. 1, a peak was not seen for unknown cis isomer 1 ("the C1 peak").Typically, this isomer will be seen after a retention time of about 14minutes. The percentages given in the above table are approximate.

The individual peak areas are determined as a percentage by dividing theindividual peak areas by the total peak area and multiplying the resultby 100 to give the percentage of total carotenoid. These percentagesappear under the heading "Conc" in the table that appears below theexample chromatogram in FIG. 1.

Alternatively, the following formula can also be used in calculating thecis isomers percentage of the total carotenoid content. The formulapresumes that a C1 peak is seen in the chromatogram:

    Cis %=(C1+C2+C3+C4/(Total peak areas))×100%

Cis isomers are not always fully resolved but this does not effect theirtotal cis isomer absorbance. In this regard, the C1 peak is not alwaysfully resolved or separated from the other cis isomers. In this case,the C1 peak can be neglected from the calculation.

The all trans isomer percentage of the carotenoid content can also bedetermined by the following formula:

    Trans %=(T/(Total peak areas))×100%

When conducting this type of analysis it is recommended that low actinicglasswater is used as beta-carotene is degraded slowly in light. Theanalysis should also be performed in duplicate because the retentiontimes are very dependant on temperature, the column and solvents shouldbe maintained at 20° C.

Accordingly, the invention provides a novel carotenoid compositionderived from a natural source, of high cis beta-carotene content.

I claim:
 1. A carotenoid composition derived from a natural source,wherein at least 50% by weight of the carotenoid content of thecomposition is cis beta-carotene.
 2. A carotenoid composition accordingto claim 1, wherein the cis beta-carotene content of the composition isbetween at least 50% and 80% by weight.
 3. A carotenoid compositionaccording to claim 1, wherein the cis beta-carotene content of thecomposition is between 60% and 70% by weight.
 4. A carotenoidcomposition according to claim 1, wherein the cis beta-carotene contentof the composition is between 60% and 85% by weight.
 5. A carotenoidcomposition according to claim 1, wherein the composition ispredominantly 9 cis beta-carotene.
 6. A carotenoid composition accordingto claim 1, wherein the composition comprises 70% 9 cis beta-carotene byweight.
 7. A carotenoid composition according to claim 5 wherein thesource of the 9 cis beta-carotene is selected from the group consistingof plant products, flowers of certain species of the Acacia genus,cucurbitaceae and Dunalliela salinea and mixtures thereof.
 8. Acarotenoid composition according to claim 5, wherein the source of the 9cis beta-carotene is Dunaliella salina.
 9. A carotenoid compositionaccording to claim 1, wherein the composition is dispersed in a naturalcarrier oil selected from the group consisting of animal oil, vegetableoil, and mineral oil, and mixtures thereof.
 10. A carotenoid compositionaccording to claim 9, wherein the oil carrier is selected from olive,corn, soya bean, essential oils, terpene based oils, fish derived oilsand mixtures thereof.
 11. A carotenoid composition according to claim 1,wherein the composition further comprises small quantities of oilsoluble anti-oxidants in the range of 0.01% to 1.0% by weight of pureanti-oxidant.
 12. A carotenoid composition according to claim 1, whereinthe composition further comprises small quantities of oil solubleanti-oxidants in the range of 0.01% to 0.5% by weight of pureanti-oxidant.
 13. A carotenoid composition according to claim 11,wherein the anti-oxidant is selected from butylated hydroxy anisole,butylated hydroxytoluene, propyl gallate, ethoxyquin and ascorbylpalmitate plus other internal antioxidant extracts which may be derivedfrom herbs, and natural tocopherols, and mixtures thereof.
 14. A methodfor producing a carotenoid composition derived from a natural source,wherein the carotenoid composition contains at least 50% by weight ofcis-beta-carotene, said method comprising(a) forming acarotenoid-containing preparation from the natural source, (b) treatingthe preparation by using density differentiation means, so as toseparate cis-isomer carotenoids from trans-isomer carotenoids containedin the preparation.
 15. The method of claim 14, wherein the densitydifferentiation means include centrifugation.
 16. A method of producinga carotenoid composition derived from the group consisting of a naturalsource, wherein the carotenoid composition contains at least 50% byweight of cis-beta-carotene, said method comprising(a) forming acartenoid-containing preparation from the natural source, (b) treatingpreparation by using the filtration means so as to separate cis-isomercarotenoids from trans-isomer carotenoids contained in the preparation.17. The method of claim 16, wherein the filtration means is selectedfrom the group consisting of a filter press, a drum filter, a filterbed, a filter cartridge system, a membrane filter or a combination ofthe foregoing.
 18. A carotenoid composition according to claim 7,wherein the plant products are apricots.
 19. A carotenoid compositionaccording to claim 13, wherein the antioxidants are natural tocopherols.